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Related Experiment Videos

Solvent accessibility of the thrombin-thrombomodulin interface.

J G Mandell1, A Baerga-Ortiz, S Akashi

  • 1Department of Chemistry and Biochemistry, University of California, San Diego, CA 92093-0359, USA.

Journal of Molecular Biology
|February 17, 2001
PubMed
Summary

Thrombin

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Biochemistry·2001

Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Interactions

Background:

  • Thrombin plays a critical role in hemostasis and thrombosis.
  • Thrombomodulin (TM) modulates thrombin's activity, regulating coagulation.
  • Understanding the structural basis of thrombin-TM interaction is crucial for therapeutic development.

Purpose of the Study:

  • To investigate the kinetics of solvent accessibility at the protein-protein interface between thrombin and thrombomodulin epidermal growth factor-like domain 45 (TMEGF45).
  • To characterize the dynamic changes in thrombin's structure upon binding to TMEGF45.
  • To correlate structural changes with functional implications in thrombin activity.

Main Methods:

  • Amide hydrogen/deuterium (H/2H) exchange monitored by MALDI-TOF mass spectrometry to assess solvent accessibility.
  • Surface plasmon resonance (SPR) to measure association and dissociation rate constants.
  • H/2H exchange rate measurements at varying pH and TMEGF45 concentrations.

Main Results:

  • Thrombin-TMEGF45 interaction is rapid and reversible.
  • Binding of TMEGF45 renders two thrombin segments, part of anion-binding exosite I, completely solvent-inaccessible.
  • Other thrombin regions show partial solvent-inaccessibility, with pH-dependent exchange rates, suggesting allosteric modulation.

Conclusions:

  • TMEGF45 binding induces significant, localized structural changes in thrombin, particularly at anion-binding exosite I.
  • These structural alterations are linked to functional modulation and allosteric effects.
  • The study provides insights into the dynamic interface of thrombin-thrombomodulin complex formation.

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